scholarly journals The broad specificity of dominant inhibitory protein kinase C mutants infers a common step in phosphorylation

1998 ◽  
Vol 333 (3) ◽  
pp. 631-636 ◽  
Author(s):  
Pilar GARCIA-PARAMIO ◽  
Yolanda CABRERIZO ◽  
Frederic BORNANCIN ◽  
Peter J. PARKER
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Dominant Negative ◽  
Activation Loop ◽  
Phosphorylation Sites ◽  
Inhibitory Protein ◽  
Kinase C ◽  
Broad Specificity

Dominant negative properties are conferred on protein kinase (PK) Cα by mutation of the phosphorylation site in the activation loop of the kinase domain. To address the universality and/or specificity of such mutations, analogous alterations were introduced in other members of the PKC family and tested for their effects on the function of co-transfected activated PKC. For all three subclasses of the PKC family, mutations of the predicted activation loop phosphorylation sites resulted in dominant negative properties. These properties were not restricted to the cognate PKC isotypes, but were effective across the different subclasses. For example, two PKCζ mutants (atypical isotype) inhibited both PKCα (classical isotype) and PKCε (novel isotype). For all these mutants, inhibition correlated with an ability to prevent the accumulation of phosphorylated PKCα, consistent with the expected mode of action. In the case of the PKCα mutant, it was shown that inhibition required the full-length mutant protein. The results provide evidence for the involvement of a common step in the phosphorylation of all PKC isotypes.

scholarly journals Alternate splicing in human Na+-MI cotransporter gene yields differentially regulated transport isoforms

1999 ◽  
Vol 276 (6) ◽  
pp. C1325-C1337 ◽  
Author(s):  
Francesca Porcellati ◽  
Yoshiyuki Hosaka ◽  
Tommy Hlaing ◽  
Masaki Togawa ◽  
Dennis D. Larkin ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Phosphorylation Site ◽  
Open Reading Frame ◽  
Phosphorylation Sites ◽  
Reading Frame ◽  
Kinase C ◽  
Multiple Protein ◽  
Kinase A

myo-Inositol is a ubiquitous intracellular organic osmolyte and phosphoinositide precursor maintained at millimolar intracellular concentrations through the action of membrane-associated Na+- myo-inositol cotransporters (SMIT). Functional cloning and expression of a canine SMIT cDNA, which conferred SMIT activity in Xenopus oocytes, predicted a 718-amino acid peptide homologous to the Na+-glucose cotransporter with a potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites. A consistent ∼1.0- to 13.5-kb array of transcripts hybridizing with this cDNA are osmotically induced in a variety of mammalian cells and species, yet SMIT activity appears to vary among different tissues and species. An open reading frame on human chromosome 21 (SLC5A3) homologous to that of the canine cDNA (96.5%) is thought to comprise an intronless human SMIT gene. Recently, this laboratory ascribed multiply sized, osmotically induced SMIT transcripts in human retinal pigment epithelial cells to the alternate utilization of several 3′-untranslated SMIT exons. This article describes an alternate splice donor site within the coding region that extends the open reading frame into the otherwise untranslated 3′ exons, potentially generating novel SMIT isoforms. In these isoforms, the last putative transmembrane domain is replaced with intracellular carboxy termini containing a novel potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites, and this could explain the heterogeneity in the regulation and structure of the SMIT.

Characterization of Protein Kinase C θ Activation Loop Autophosphorylation and the Kinase Domain Catalytic Mechanism

Biochemistry ◽  
2005 ◽  
Vol 44 (28) ◽  
pp. 9563-9573 ◽  
Author(s):  
Robert Czerwinski ◽  
Ann Aulabaugh ◽  
Rita M. Greco ◽  
Stephane Olland ◽  
Karl Malakian ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Catalytic Mechanism ◽  
Kinase Domain ◽  
Activation Loop ◽  
Kinase C

scholarly journals Dynamics of Protein Kinase C-mediated Phosphorylation of the Complement C5a Receptor on Serine 334

2006 ◽  
Vol 282 (7) ◽  
pp. 4345-4353 ◽  
Author(s):  
Beatrix Pollok-Kopp ◽  
Friederike Hüttenrauch ◽  
Stephanie Rethorn ◽  
Martin Oppermann
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Phosphorylation Sites ◽  
Recombinant Enzymes ◽  
C5a Receptor ◽  
Kinase C ◽  
Complement C5a ◽  
C5a Anaphylatoxin

Upon agonist binding, the C5a anaphylatoxin receptor (C5aR) is rapidly phosphorylated on phosphorylation sites that are located within the C-terminal domain of the receptor. Previous studies suggested that C5aR phosphorylation proceeds in a hierarchical manner with serine 334 presenting a highly accessible priming site that controls subsequent phosphorylation at other positions. To better understand the dynamics of Ser-334 phosphorylation, we generated site-specific monoclonal antibodies that specifically react with phosphoserine 334. In differentiated U937 cells, which endogenously express C5aR, stimulation with low C5a concentrations resulted in a very rapid (t½ ∼ 20 s), albeit transient, receptor phosphorylation. Whole cell phosphorylation assays with specific inhibitors as well as in vitro phosphorylation assays with recombinant enzymes and peptide substrates revealed that phosphorylation of Ser-334 is regulated by protein kinase C-β and a calyculin A-sensitive protein phosphatase. Surprisingly, at high concentrations (>10 nm) of C5a, the protein kinase C-mediated phosphorylation of Ser-334 was essentially blocked. This could be attributed to the even faster (t½ < 5 s) binding of β-arrestin to the receptor. Analysis of C5aR Ser/Ala mutants that possess a single intact serine residue either at position 334 or at neighboring positions 327, 332, or 338 revealed functional redundancy of C-terminal phosphorylation sites since all 4 serine residues could individually support C5aR internalization and desensitization. This study is among the first to analyze in a detailed manner, using a non-mutational approach, modifications of a defined phosphorylation site in a G protein-coupled receptor and to correlate these findings with functional parameters of receptor deactivation.

scholarly journals A potent and highly selective peptide substrate for protein kinase C assay

1997 ◽  
Vol 322 (2) ◽  
pp. 455-460 ◽  
Author(s):  
Reet TOOMIK ◽  
Pia EK
Keyword(s):  
Amino Acids ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Phosphorylation Site ◽  
Phosphorylation Sites ◽  
Cell Extracts ◽  
Kinase C ◽  
Km Value ◽  
Peptide Phosphorylation

Protein kinases exhibit substrate specificities that are often primarily determined by the amino acids around the phosphorylation sites. Peptides corresponding to protein kinase C phosphorylation sites in several different proteins were synthesized on SPOTs membrane which has recently been found to be applicable for studies of protein kinase specificity. After phosphorylation with protein kinase C, we chose the best phosphorylated peptides for the investigation of the importance of amino acids immediately adjacent to the phosphorylation site. The selectivity of the best protein kinase C substrates from this study was analysed with protein kinases A, CK1 and CK2. According to these tests, the most favourable characteristics of SPOTs-membrane-associated peptides were demonstrated by peptide KRAKRKTAKKR. Kinetic analysis of peptide phosphorylation with protein kinase C revealed an apparent Km of 0.49±0.13 μM and Vmax of 10.0±0.5 nmol/min per mg with soluble peptide KRAKRKTAKKR. In addition, we assayed several other soluble peptides commonly used as protein kinase C substrates. Peptide KRAKRKTAKKR showed the lowest Km and the highest Vmax/Km value in comparison with peptides FKKSFKL, pEKRPSQRSKYL and KRAKRKTTKKR. Furthermore, of the peptides tested, KRAKRKTAKKR was the most selective substrate for protein kinase C. The favourable kinetic parameters combined with the selectivity should make the KRAKRKTAKKR peptide useful as a substrate for protein kinase C in the assays of both purified enzyme and in crude cell extracts.

Regulation of novel protein kinase C ε by phosphorylation

2002 ◽  
Vol 363 (3) ◽  
pp. 537-545 ◽  
Author(s):  
Vittoria CENNI ◽  
Heike DÖPPLER ◽  
Erica D. SONNENBURG ◽  
Nadir MARALDI ◽  
Alexandra C. NEWTON ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Intracellular Localization ◽  
Kinase Domain ◽  
Activation Loop ◽  
3T3 Fibroblasts ◽  
Kinase C ◽  
Hydrophobic Motif ◽  
Nih 3T3

The activity and intracellular localization of protein kinase C (PKC) family members are controlled by phosphorylation at three highly conserved sites in the catalytic kinase domain. In the case of the novel PKC∊ isoform, these are Thr566 in the activation loop, Thr710 in the turn motif and Ser729 in the C-terminal hydrophobic motif. In the present study, we analysed the contribution of the phosphoinositide-dependent kinase 1 (PDK-1) and PKC∊ kinase activity in controlling the phosphorylation of Thr566 and Ser729. In NIH 3T3 fibroblasts, PKC∊ migrated as a single band, and stimulation with platelet-derived growth factor resulted in the appearance of a second band with a slower electrophoretic mobility, concomitant with an increase in phosphorylation of Thr566 and Ser729. Cells transfected with an active PDK-1 allele also resulted in increased PKC∊ Thr566 and Ser729 phosphorylation, whereas an active myristoylated PKC∊ mutant was constitutively phosphorylated at these sites. Protein kinase-inactive mutants of PKC∊ were not phosphorylated at Ser729 in cells, and phosphorylation of this site leads to dephosphorylation of the activation-loop Thr566, an effect which can be reversed with either okadaic acid or co-transfection with active PDK-1. In vitro, PDK-1 catalysed the phosphorylation of purified PKC∊ in the presence of mixed micelles containing either diacylglycerol or PtdIns(3,4,5)P3, concomitant with an increase in Ser729 phosphorylation. These studies reveal that the mechanism of phosphorylation of a novel PKC is the same as that for conventional PKCs: PDK-1 phosphorylation of the activation loop triggers autophosphorylation of the hydrophobic motif. However, the regulation of this phosphorylation is different for novel and conventional PKCs. Specifically, the phosphorylation of novel PKCs is regulated rather than constitutive.

scholarly journals Role of the kinase activation loop on protein kinase C θ activity and intracellular localisation

FEBS Letters ◽  
2003 ◽  
Vol 554 (1-2) ◽  
pp. 35-40 ◽  
Author(s):  
Bianca Sparatore ◽  
Mario Passalacqua ◽  
Marco Pedrazzi ◽  
Sabina Ledda ◽  
Mauro Patrone ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Activation Loop ◽  
Kinase Activation ◽  
Kinase C ◽  
Intracellular Localisation
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